Thousands of labs worldwide are studying helical membrane proteins. These proteins regulate the trafficking of water, ions, and other molecules into and out of the cell.
A host of human diseases are caused by defects in membrane proteins. These proteins are highly hydrophobic molecules that reside in the fatty layer that surrounds all human cells, and control the flow of the materials vital to life in and out of the cell, cellular growth, and regulation. Despite the importance of these molecules in maintaining the health of the human body, research on membrane proteins is in its relative infancy versus their water-soluble protein counterparts. In particular, the study of membrane proteins has been hindered by their insolubility when extracted outside their native membrane environment—a necessary step in the characterization of their function and malfunction. These challenges are evidenced by the extended gaps in the discovery-to-therapeutic pipeline for diseases traceable to membrane protein defects. Cystic fibrosis, for example, is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR)—a membrane protein—yet remains incurable 20 years after the discovery of the CFTR gene, in large part because technologies appropriate for detailed study of this and other membrane proteins in the laboratory have not been available. Therefore, new research tools specific to the specific physiochemical properties of membrane proteins must be developed.
Since membrane proteins occur largely on the surfaces of cells, they are the major targets accessible to drug action. The relevance of these molecules in drug development is reflected in the large number of therapeutics on the market directed at membrane proteins, which accounted for about 70% of the pharmaceuticals approved by the FDA from 1996-2006. G-protein coupled receptors, for example, have been termed the ‘staple diet’ of the pharmaceutical industry. It has nevertheless been suggested that certain membrane protein families are relatively under-explored in terms of drug discovery. Membrane protein research thus represents a broad opportunity for the pharmaceutical industry to expand its range of target molecules.
Virtually all human diseases, whether inherited or acquired, are caused when the protein(s) responsible for an important biological activity fail to perform their function. The involvement of membrane proteins in virtually all cellular mechanisms of survival and reproduction make them crucial research targets in the understanding of these mechanisms and the diseases they can engender. Yet, new drugs against such disease-causing molecules in the body cannot be developed until the target molecules have first been identified and characterized.
Membrane proteins represent fully one-third of the human genome. They are key action macromolecules in the human body, serving as enzymes, nutrient transporters, signalling systems, and as participants in a myriad of activities involving vision, smell, taste, cognition, memory, and motion. It is now known that defects or deficiencies in membrane proteins underlie a striking array of human diseases, including, but not limited to, cystic fibrosis, neurological disorders, diabetes, Alzheimer's, multiple sclerosis, muscular dystrophy, heart and kidney diseases, many forms of cancer, and lethal genetic diseases. Membrane proteins are also intermediaries in various modes of bacterial drug resistance in infectious disease, and serve as receptors for infection by viruses such as HIV. Afflictions such as addiction, cognition and memory, depression, and schizophrenia have all been associated with membrane proteins. More than ever, if biological science is to successfully treat human diseases that have thus far evaded our boldest efforts, elucidation of the basic mechanisms that underlie human disease, and how/why these protein molecules become compromised in disease, is an absolute necessity.
Importantly, the challenges inherent in membrane protein production, isolation, identification, and stability are currently yielding to modern molecular biological techniques, and researchers in this field are now poised to make important advances in their understanding of the mechanisms of action of these vital proteins.
Membrane proteins are distinguished from water-soluble proteins by their highly hydrophobic character which necessitates their maintenance in detergents such as sodium dodecylsulfate (SDS) or non-denaturing detergents such as Triton-X100 or dodecylmaltoside for study. The structure of the S. lividans KcsA potassium channel exemplifies membrane protein topology. The hydrophobic portions of TM helices are flanked by positively charged residues and/or aromatic residues and are separated by hydrophilic and/or polar loop regions. This amino acid distribution illustrates the general layout of the TM segments of helical membrane proteins.
Proteins of each type can be routinely and productively examined for their purity, size, and stoichiometry on a protein sizing technique known as sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This procedure, arguably the most commonly used laboratory technique in the world, compares the gel migration distance of the protein(s) of interest to that of commercially available water-soluble protein calibration standards in order to determine molecular weight (MW). While SDS-PAGE typically estimates the sizes of water-soluble proteins with reasonable accuracy, estimates of membrane protein MWs are commonly inaccurate.
These sizing discrepancies can arise from an increased amount of SDS detergent bound to denatured membrane proteins versus the water-soluble polypeptides used for gel calibration, and may also arise as a result of the amount of non-denatured structure in membrane proteins. This work clearly indicates that the commercially available water-soluble protein MW standards universally used to estimate protein size on SDS-PAGE are inappropriate for use with membrane proteins. Accordingly, there is a need to develop a tool to assist in the characterization, purification, and estimation of structural stability of membrane proteins.